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British Journal of Anaesthesia 1990; 64: 723-727



KEY WORDS Anaesthetics volatile: halothane, isoflurane. Blood: chemotaxis, leucocytes, neutrophils.


The methods utilized were approved by the Ethics and Research Committee of the University of Cape Town. An important characteristic of phagocytic leucoNeutrophils were elicited into the peritoneal cytes is their ability to move towards a chemotactic cavity of adult New Zealand Cross rabbits. Fifty stimulus. This response is an essential part of host millilitre of 5 % casein in 0.9 % saline was injected defence against infection. Moudgil and colleagues into the rabbit's peritoneal cavity. Twenty-four [1] showed that halothane, trichloroethelene, hours later, the peritoneal cavity was washed out diethyl ether and methoxyflurane inhibited the with 150 ml of a modified Ringer's solution, in ability of human neutrophils to move towards the which the lactate had been replaced by an chemoattractant agent, casein. In 1984 it was equivalent molar concentration of bicarbonate shown [2] that isoflurane did not affect neutrophil (Plasmalyte B). The rabbits were anaesthetized chemotaxis, whereas nitrous oxide, methoxy- for each procedure utilizing halothane in oxygen flurane, halothane and enflurane delivered at 1 via a Magill system. Each procedure took 10 min. MAC depressed the ability of human neutrophils The harvest of cells contained 2-15 x 10" cells, to move towards casein. 92-96 % of which were neutrophils. Some macroIt is accepted that inhalation anaesthetic agents phages were obtained also in these washings. depress various aspects of the immune response. Cells were pelleted by spinning the suspension As part of an investigation of newer i.v. an- in a centrifuge at 110 g for 10 min. They were aesthetic agents on aspects of the immune system, resuspended in a solution containing RPMI cell we first investigated halothane and isoflurane in culture medium and 5 % human stabilized order to validate a model. We investigated the serum (2:1) at 37 °C. The dilution was adjusted effects of isoflurane and halothane on the di- to give afinalcell count of 1.5 x 10" cells ml"1. Ten rectional movement of elicited rabbit neutrophils; millilitre of the cell suspension was placed in three neutrophils elicited into the peritoneal cavity have been studied widely and are a convenient source of cells. It was felt that activated neutrophils RICHARD ERSKINE, F.F.A.(S.A.); M. F. M. JAMES, F.F.A.R.CS. ; would be more appropriate than quiescent cells, Department of Anaesthesia, University of Cape Town, Medical School, Anzio Road, Observatory 7925, S. Africa. as they would approximate more closely to cells Accepted for Publication: December 7, 1989. activated by the stress response. Preliminary Correspondence to R.E.

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We have studied the effect of isoflurane and halothane on the chemotactic function of elicited rabbit neutrophi/s in vitro . In comparison with neutrophils not exposed to each volatile agent, isoflurane stimulated (? = 3.5 x 10~5), but halothane had no effect on the movement of cells towards the chemical attractant, casein.

studies in this model with 2 % halothane appeared to confirm the findings of Moudgil and colleagues [1] of substantial inhibition of movement through the niters. In view of the difficulty of conducting these experiments on the same rabbit cell lines at different concentrations of volatile agents, we investigated the effects of the agents in concentrations likely to be used clinically—approximately 1.5 MAC.


724 27Z Oxygen 68 Z Nitrogen 5Z Carbon dioxide \

EMMA Test tube .•—containing cell suspension



FIG. 1. Apparatus used for bubbling the agents through the neutrophils. V = Vaporizer; X = 8 a t e control clamp. Anaesthetic gas inlet


Syringe .

— Frame Cell suspension Casein.

Micropore filter

FIG. 2. Apparatus used to incubate the neutrophils in the absence or presence of casein. Only one of 18 syringe barrels in each box is illustrated.

15-ml plastic test tubes at 37 °C. A gas mixture of 5% carbon dioxide, 30% oxygen and nitrogen together with 1 % halothane, 1.6 % isoflurane or no vapour was bubbled through the cells for 20 min, which is similar to the time shown in previous studies to be necessary for equilibration. The gases were bubbled through the cell suspension via a 23-gauge needle at a rate of approximately 60 bubbles per minute. The vapours were delivered from a Drager Vapor vaporizer for halothane and a Fortec Mark III

vaporizer for isoflurane. Vapour partial pressures were checked using an EMMA Engstrom vapour analyser. The accuracy of the internal calibration of the EMMA had been checked previously against standard partial pressures of the volatile agents made up by vaporizing a known mass of volatile agent into a flask of known volume at measured temperature and pressure. In order to ensure adequate flow through each vaporizer, flows in excess of 1 litre min"1 were passed through each vaporizer. Low flows distal to the

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Air Oj CO2











1.8 2 0

2 2 2.4 2.6

FIG. 3. Typical example of cell counts obtained in one rabbit cell line in a single experiment at various concentrations of casein. Mean (1 SD) of 10 counts per high power (HP) field at each point. Note the decline in neutrophil movement with increasing concentrations of casein greater than 0.6%. O = Control; • = halothane; A = isoflurane.

vaporizer were delivered to the test tubes using Yconnections with gate clamps adjusted to produce a steady, slow bubbling through the cell suspension (fig. 1). Cell movement towards the chemoattractant, casein, was assessed using Wilkinson's modification of Boyden chambers [3]. After the initial equilibration with the anaesthetic agents, 200-|il aliquots of cell suspension were placed above 5-|im pore Millipore filters in sawn-off tuberculin syringe barrels. The filters had been attached previously to the syringe barrels with UHU glue, an ethanol soluble adhesive which has no effect on neutrophil function. The lower chamber (10-ml glass beakers) contained concentrations of 0, 0.2, 0.4,0.6, 1.2 and 2.4 % casein in 0.9 % saline. Two hundred microlitre of the 5 % human stabilized serum was added to the bottom chamber in the beaker which contained no casein. The experiments were conducted in three plastic sandwich boxes with tight fitting lids, with small inlet and outlet ports for the anaesthetic gases. Efflux from the boxes also was monitored using the Engstrom EMMA (fig. 2). Each cell suspension received the same anaesthetic as before. After 60 min of incubation, the cell migration through the niters was stopped by immersion of the barrels in ethanol. This also dissolved the glue and allowed the detached niters to be stained in haematoxylin using a standard technique. Cell movement through the filters was assessed by counting the number of cells per high power field that had traversed the filter paper completely and remained

attached to its under surface. Ten fields from two filter papers were counted for each concentration of casein. We found that cell migration diminished at concentrations of casein greater than 0.4 % in the presence of both volatile agents and in the control cells. With 2.4% casein there was no difference in movement compared with that in the absence of casein (fig. 3). This effect was apparent in all the rabbit cell preparations and has been shown previously with rabbit neutrophils [4]. We therefore discarded the results from 2.4% casein in all groups. In order to differentiate between caseinactivated and random neutrophil motion, the results were divided into those in which no casein was present and the combined results from 0.2-1.2% casein concentrations. The effects of halothane and isoflurane were compared with controls in each experiment by calculating the percentage difference from the movement exhibited in the control experiment. Results obtained in the halothane group were compared with those in the isoflurane group using a Wilcoxon rank sum test. RESULTS

The pooled results of our experiments from five rabbits are shown in figure 4. Halothane 1 % did not depress the movement of neutrophils towards casein, whilst isoflurane produced significant stimulation of movement of neutrophils through the filter papers (P = 3.5 x 10"6).

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Casein concentration (Z)



increased movement towards the chemoattractant casein in the presence of 1.6% isoflurane. No effect was present when 1 % halothane was administered to the same cells. 600 Several studies have attempted to resolve the effects of inhaled anaesthetic agents on neutrophil motile function. Bruce [5] showed that halothane x inhibited peritoneal neutrophil mobilization folX lowing injection of S. typhimurium into the o peritoneal cavity of mice, and suggested that X •fc 200 general anaesthesia may depress natural defences X to infection. Moudgil and his colleagues [1] showed reduced chemotactic responses in human neutrophils exposed to various inhalation agents, including halothane, and subsequently confirmed this effect using modern agents at 1 MAC I I -200 concentration [2]. In ascending order of effect, No casein Casein enflurane, halothane, methoxyflurane and 70% FIG. 4. Pooled results from five rabbit cell lines exposed to nitrous oxide inhibited neutrophil chemotaxis. either halothane (O) or isoflurane (x) expressed as a differIsoflurane was the only agent tested in this model ence from the control cells. There is no difference between which did not depress chemotaxis. In contrast, the cell counts obtained with no attractant present. Cells exposed to casein exhibited increased movement over control Duncan and Cullen [6] were unable to demcells in the isoflurane group (P = 1 x 10"5). Cells exposed to onstrate any effect of halothane, although they halothane did not exhibit any difference in movement over used a chemoattractant different from the casein their controls. The difference in movement between cells exused by Moudgil. With casein as the attractant, posed to isoflurane and those exposed to halothane is highly Nunn and colleagues [7] were not able to significant statistically (P = 3.5 x 10~6). demonstrate any depressant effect of halothane on random movement, chemotaxis or phagocytosis in the human neutrophils which they studied, alDISCUSSION Conflicting results have been reported in previous though a subsequent study showed marked destudies of the effects of volatile agents on neutro- pression of chemotaxis by nitrous oxide [8]. Our phil movement (table I). We have demonstrated results support those which were unable to show that elicited rabbit neutrophils in vitro exhibit depression of chemotaxis with halothane, but it is 800

TABLE I. Summary of studies of the effect of inhalation anaesthetic agents on neutrophil movement, presented in chronological sequence. ± Increased = Tendency to increase, but not significant Agent



2% Halothane 0.75 % Halothane 1.15% Isoflurane 1.68% Enflurane 70 % Nitrous oxide

Human Human Human Human Human Human Human Human Human Human

0.01-0.1 % Casein 0.01-0.1 % Casein C,A C,A C.A 0.5 % Casein 0.05-0.2% 0.05-0.2% 0.05-0.2% 0.05-0.2%

70 % Nitrous oxide 80 % Nitrous oxide 1.0% Halothane 1.6% Isoflurane

Human Human Elicited rabbit

0.1% Casein 0.1% Casein 0.2-2.4% Casein 0.2-2.4% Casein

1 % Halothane 2% Halothane 0.5% Halothane 1.0% Halothane 2.0% Halothane

Casein Casein Casein Casein

Random movement

Chemotaxic movement


Decreased Decreased ± Increased No change No change

Decreased Decreased No change ±Increased ±Increased

Increased Decreased No change No change

No change Decreased No change Decreased

Nunn [7] Moudgil [2]

No No No No

Decreased + + Decreased + + No change Increased

Nunn [8]

change change change change

Moudgil [1] Duncan [6]

Present study

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These functions have been tested also in in vitro cell suspensions in the presence of volatile agents. Here also, workers have produced conflicting results. The changes in cell function in the presence of volatile agents appear to be marked, although also very variable. It is only when we understand the mechanisms whereby volatile agents affect neutrophil function that it will be possible to explain the present conflicting results. REFERENCES 1. Moudgil GC, Allan RB, Russel RJ, Wilkinson PC. Inhibition, by anaesthetic agents, of human leucocytic locomotion towards the chemical attractants. British Journal of Anaesthesia 1977; 49: 97-104. 2. Moudgill GS, Gordon J, Forrest JB. Comparative effects of volatile anaesthetic agents and nitrous oxide on human leucocytic chemotaxis in vitro. Canadian Anaesthetists Society Journal 1984; 31: 631-637. 3. Wilkinson PC. Locomotion and chemotaxis of leucocytes. In: Weir DM, Herzenberg LA, Blackwell C, Herzenberg L, eda. Handbook of Experimental Immunology. Book II: Cellular Immunology. Oxford: Blackwell Scientific Publications, 1986; 51.1-51.6. 4. Todd G. An in vitro study of neutrophil chemotaxis. [Ph.D. Thesis]. Cape Town: University of Cape Town, 1976. 5. Bruce DL. Effect of halothane anesthesia on experimental salmonella peritonitis in mice. Journal of Surgical Research 1967; 7: 180-185. 6. Duncan PG, Cullen BF. Neutrophil chemotaxis and anaesthesia. British Journal of Anaesthesia 1977; 49: 345-351. 7. Nunn JF, Sturrock JE, Jones AJ, O'Morain C, Segal AW, Coade SB, Doring J, Walker D. Halothane does not inhibit human neutrophil function m vitro. British Journal of Anaesthesia 1979; 51: 1101-1108. 8. Nunn JF, O'Morain C. Nitrous oxide decreases motility of human neutrophils in vitro. Anesthesiology 1982; 56: 45-48.

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not clear why there is this discrepancy between various studies. Moudgil [1,2] used a much smaller concentration of casein than that used by ourselves or by Nunn and colleagues [7], and it is possible that his results reflect a relatively subtle effect of halothane, which is masked in higher concentrations of casein. Support for this hypothesis may be found in the agreement of both groups of workers that nitrous oxide has a depressant effect, which was greater than that of the other agents in MoudgiPs study. It is also of interest that, in Nunn's study showing a depressant effect of nitrous oxide [8], a low concentration (0.1 %) of casein was used. The reason for the enhanced movement in the presence of isoflurane cannot be deduced from these experiments. Increase in chemotactic movement of neutrophils may result from several factors, including increased sensitivity of chemotactic recognition, changes in cell membrane receptor sites, or increased mobilization of the microfilamentous locomotor system. We are undertaking further studies to try to identify the mechanisms that may be responsible for these results. A possible contributory factor may be our use of cells which had already been activated by elicitation into the rabbit's peritoneal cavity. If the cells were thus rendered more motile, this might also explain partially the absence of effect from halothane, especially if this action is a subtle one. A species difference also cannot be excluded. Movement of neutrophils towards a chemical stimulus is only one of the functions which the cell performs in ingesting and killing bacteria. The release of active oxygen, ingestion of bacteria and killing of bacteria are also vital functions.